Published on April 2017 | Ophthalmic Genetics

Identification and characterization of variants and a novel 4 bp deletion in the regulatory region of SIX6, a risk factor for primary open‐angle glaucoma

Background Primary open‐angle glaucoma (POAG) is a complex disease of multigenic inheritance and the most common subtype of glaucoma. SIX6 encodes a transcription factor involved in retina, optic nerve, and pituitary development. Previous studies showed a genetic association between the SIX6 locus and POAG, identifying risk alleles. Whether these alleles are present also in the south Indian population is unclear. Methods To address this question, the SIX6 gene and an already characterized and highly conserved SIX6 enhancer (Ch14:60974427‐60974430) were sequenced in two south Indian cohorts, respectively, composed of 65/65 and 200/200 POAG cases/age‐matched controls. We next used Taqman‐based allelic discrimination assay to genotype a common variant (rs33912345: c.421A>C) and the rs1048372 SNP in two cohorts, respectively, composed of 557/387 and 590/448 POAG cases/age‐matched controls. An additional cohort of 153 POAG cases was subsequently recruited to assess the association of the rs33912345:c.421A>C and rs10483727 variants with more prominent changes in two POAG diagnostic parameters: retinal nerve fiber layer thickness and vertical cup/disc ratio, using spectral domain optical coherence tomography. The activity of the newly identified enhancer variants was assessed by transgenesis in zebrafish and luciferase assays. Results We identified two known rare and two common variants in the SIX6 locus and a novel 4 bp deletion in the analyzed enhancer. Contrary to previous studies, we could not establish a significant association between the rs10483727 and rs33912345:c.421A>C variants and PAOG in the south Indian ethnicity but patients carrying the corresponding C or T risk alleles exhibited a dose‐dependent reduction of the thickness of the retinal nerve fiber layer and a significant increase in the vertical cup/disc ratio. Transgenesis in zebrafish and luciferase assays demonstrated that the newly identified 4 bp deletion significantly reduced reporter expression in cells of the retinal ganglion and amacrine layers, where human SIX6 is expressed. Conclusion Altogether, our data further support the implication of SIX6 variants as POAG risk factors and implicates SIX6 haploinsufficiency in POAG pathogenesis.